Method for purification of alpha-1-antitrypsin
Inventors
Kee, Scott M. • Cook, Paul I. • Smith, James R. • Kling, Robert • Fowler, Scott A. • Weber, David
Assignees
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Publication Number
US-7777006-B2
Publication Date
2010-08-17
Expiration Date
Abstract
A streamlined method for purifying alpha-1-antitrypsin (AAT) from an AAT-containing protein mixture, such as a Cohn fraction IV precipitate, is provided. In the method of the invention, contaminating proteins are destabilized by cleavage of disulfide bonds with a reducing reagent, such as a dithiol, which does not affect AAT. The destabilized proteins are then preferentially adsorbed on a solid protein-adsorbing material, without the addition of a salt as a precipitant. Separation of the solid adsorbent from the solution leaves a purified AAT solution that is directly suitable for chromatographic purification, without the need for extensive desalting as in prior art processes. A process incorporating this method, which provides pharmaceutical grade AAT in high yield on a commercial scale, is also described.
Core Innovation
The invention provides a method for purifying alpha-1-antitrypsin (AAT) from an AAT-containing protein mixture or from an AAT-containing Cohn fraction IV protein precipitate. The method reduces disulfide bonds using a disulfide-reducing agent to produce a reduced AAT-containing protein mixture or reduced suspension, and then contacts the reduced mixture or suspension with a silica adsorbent to preferentially adsorb contaminating proteins. The AAT is isolated to obtain a clarified AAT solution without using salting out.
A key aspect of the invention is the use of silica adsorbent materials in combination with disulfide reduction, while explicitly excluding any step of the method being a salting out step. The invention further characterizes the purification workflow by including anion exchange chromatography and hydrophobic interaction chromatography to remove contaminating proteins, including contaminating proteins that are more hydrophobic than AAT.
The invention additionally frames production of pharmaceutical-grade AAT by incorporating viral reduction steps and formulation-related targets as described in the provided summary. Viral reduction can include pasteurization and ultrafiltration, and in some embodiments includes ultrafiltration/diafiltration. The document also describes target specifications including purity/impurity levels, functional specific activity, viral log reduction levels, and stability.
Claims Coverage
The document provides six independent claims directed to AAT purification using disulfide reduction plus silica adsorption without salting out, and in multiple independent claims further includes downstream chromatography and viral reduction steps. Across the independent claims, the core inventive features are disulfide reduction, contact with a silica adsorbent, and explicit omission of salting out; additional independent claims further add specific upstream material (Cohn fraction IV precipitate), chromatography steps (anion exchange and hydrophobic interaction), and viral reduction operations (pasteurization and ultrafiltration).
Disulfide reduction followed by silica adsorption without salting out
Contacting an AAT-containing protein mixture with a disulfide-reducing agent to produce a reduced AAT-containing protein mixture; contacting the reduced AAT-containing protein mixture with a silica adsorbent material; isolating the resulting AAT, wherein no step of the method is a salting out step.
Cohn fraction IV-based reduced suspension with silica adsorption and chromatography without salting out
Suspending an AAT-containing Cohn fraction IV protein precipitate in a buffer under conditions that permit the AAT to be dissolved; contacting the resulting AAT-containing suspension with a dithiol to produce a reduced suspension; contacting the reduced suspension with a silica adsorbent; removing the silica adsorbent with insoluble materials from the reduced suspension to obtain an AAT-containing protein solution; subjecting the AAT-containing protein solution to anion exchange chromatography wherein the AAT is first bound to the column and then selectively eluted from the column; subjecting the resulting AAT eluate to hydrophobic interaction chromatography to remove contaminating proteins which are more hydrophobic than AAT; wherein no step of the method is a salting out step.
Dissolved AAT precipitate reduction with silica adsorption without salting out
Suspending an AAT-containing protein precipitate in a buffer under conditions that permit the AAT to be dissolved; contacting the AAT-containing suspension with a disulfide-reducing agent to produce a reduced AAT-containing suspension; contacting the reduced AAT-containing suspension with a silica adsorbent; isolating the resulting AAT, wherein no step of the method is a salting out step.
Cohn fraction IV purification using dithiothreitol, fumed silica, chromatography, viral reduction, and no salting out
Suspending an AAT-containing Cohn fraction IV precipitate in a buffer under conditions that permit the AAT to be dissolved; contacting the resulting AAT-containing suspension with dithiothreitol to produce a reduced suspension; contacting the reduced suspension with a fumed silica adsorbent; removing the silica adsorbent with insoluble materials from the resulting suspension to obtain an AAT-containing protein solution; subjecting the AAT-containing protein solution to anion exchange chromatography whereby the AAT is first bound to the column and then selectively eluted from the column; subjecting the resulting AAT eluate to hydrophobic interaction chromatography to remove contaminating proteins which are more hydrophobic than AAT; subjecting the AAT-containing product of the hydrophobic interaction chromatography to one or more viral reduction steps; isolating the resulting AAT, wherein no step of the method is a salting out step.
Hydrophobic interaction chromatography followed by pasteurization and ultrafiltration without salting out
Subjecting said mixture of AAT and contaminating proteins to hydrophobic interaction chromatography to remove said contaminating proteins which are more hydrophobic than AAT, wherein the AAT and contaminating protein mixture comprises less than 0.1% albumin or transferrin; subjecting the AAT-containing product of said hydrophobic interaction chromatography of step (a) to viral reduction steps of pasteurization and ultrafiltration; wherein no step of the method is a salting out step.
Viral contaminant removal from AAT solution using pasteurization and ultrafiltration without salting out
Subjecting said solution of alpha-1-antitrypsin (AAT) to pasteurization at about 60° C. to about 70° C.; subjecting the solution to ultrafiltration; wherein the ultrafiltration is by filtration through 15-70 nm ultrafilters, and wherein no step of the method is a salting out step.
Across the independent claims, the coverage centers on purifying AAT by reducing disulfide bonds and then contacting the reduced AAT-containing mixture or suspension with a silica adsorbent, while expressly excluding any salting out step. Several independent claims further specify that AAT purification proceeds through anion exchange chromatography and hydrophobic interaction chromatography and includes viral reduction using pasteurization and ultrafiltration, with additional constraints such as Cohn fraction IV inputs and fumed silica/dithiothreitol in more specific embodiments.
Stated Advantages
Reducing the need for extensive desalting/dialysis as characterized in the provided summary.
Enabling higher protein concentrations as characterized in the provided summary.
Compatibility with downstream chromatographic purification as characterized in the provided summary.
Documented Applications
Production and purification of pharmaceutical-grade AAT using the described workflow, including downstream chromatographic purification and viral reduction/inactivation steps, as characterized in the provided summary.
Purification of AAT from an AAT-containing protein mixture as a general AAT purification use case.
Purification of AAT from an AAT-containing Cohn fraction IV protein precipitate as a specific AAT manufacturing input.
Purifying a mixture of AAT and contaminating proteins by removing more hydrophobic contaminating proteins and then applying viral reduction.
Removing viral contaminants from a solution of AAT by pasteurization and ultrafiltration.
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